Screening of anti-thrombin active components from Ligusticum chuanxiong by affinity-ultrafiltration coupled with HPLC-Q-Orbitrap-MSn.

INTRODUCTION Ligusticum chuanxiong ('chuanxiong') is a traditional Chinese medicine for promoting blood circulation and removing blood stasis, which is often used to treat thrombotic diseases. However, its potential anticoagulant active ingredients have been unexplored. OBJECTIVES The study aims to establish an affinity ultrafiltration mass spectrometry (AUF-MS) method for rapid screening of anti-thrombin active components of chuanxiong and to verify it in vitro. METHOD In this study, the chemical constituents of different parts of chuanxiong were determined. A method for rapid screening of anticoagulant active ingredients by AUF-MS was established using thrombin as an affinity receptor target. Subsequently, the anticoagulant effect of such ligands was verified by in vitro anticoagulation experiments such as chromogenic substrate method and in vitro coagulation assay. Then the possible interaction mechanism between these ligands and thrombin was further studied by molecular docking. RESULTS Twenty-one components were detected from different parts of chuanxiong. And three potential anti-thrombin active components were screened: ferulic acid, chlorogenic acid, isochlorogenic acid A by AUF coupled with high-performance liquid chromatography-quadrupole-Orbitrap mass spectrometry (HPLC-Q-Orbitrap-MSn ). The in vitro activity experiments and molecular docking revealed that these potential ligands exhibited strong binding ability and inhibitory activities on thrombin. CONCLUSION The present study revealed that chuanxiong is a traditional Chinese medicine with excellent anticoagulation effects. Meanwhile, the integrated strategy based on AUF-MS, in vitro experiments and molecular docking also provided a powerful tool for further exploration of active ingredients responsible for the anticoagulant activity in chuanxiong.

[1]  Dongya Qin,et al.  Identification of the functional food ingredients with antithrombotic properties via virtual screen and experimental studies. , 2021, Food chemistry.

[2]  Bill X. Huang,et al.  Current advances in screening for bioactive components from medicinal plants by affinity ultrafiltration mass spectrometry. , 2018, Phytochemical analysis : PCA.

[3]  U. Desai,et al.  Novel heparin mimetics reveal cooperativity between exosite 2 and sodium-binding site of thrombin. , 2018, Thrombosis research.

[4]  Jun-Rong Du,et al.  Z-ligustilide extracted from Radix Angelica Sinensis decreased platelet aggregation induced by ADP ex vivo and arterio-venous shunt thrombosis in vivo in rats. , 2009, Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan.

[5]  Wei Zhang,et al.  Ligustilide induces vasodilatation via inhibiting voltage dependent calcium channel and receptor-mediated Ca2+ influx and release. , 2006, Vascular pharmacology.

[6]  Hong Yang,et al.  Ligustilide inhibits vascular smooth muscle cells proliferation. , 2006, European journal of pharmacology.

[7]  G Klebe,et al.  Three-dimensional quantitative structure-activity relationship analyses using comparative molecular field analysis and comparative molecular similarity indices analysis to elucidate selectivity differences of inhibitors binding to trypsin, thrombin, and factor Xa. , 1999, Journal of medicinal chemistry.

[8]  S. Kobayashi,et al.  Chemical structure-activity of cnidium rhizome-derived phthalides for the competence inhibition of proliferation in primary cultures of mouse aorta smooth muscle cells. , 1993, Japanese journal of pharmacology.